Synthesis and properties of a novel brominated oligomycin A derivative

Oligomycins belong to the class of highly functionalized macrolide antibiotics that contain the hydroxyl groups, the lactone and spiro moieties, as well as the double bonds. The mechanism of cytocidal activity of oligomycins involves the inhibition of oxidative phosphorylation by preventing ATP synthesis. Design of new compounds on oligomycin A scaffold is of interest as ATP synthase inhibitors are drug candidates for the treatment of bacterial infections and cancer.¹ Recently, we reported the examples of modification at the C₇ carbonyl group and the C₂=C₃ double bond of the antibiotic.² In search of selective methods to modify oligomycin A (1), we carried out bromination of (1) with NBS in DMF at neutral pH; this procedure yielded a mono-bromo derivative (2) in 45% yield (Figure 1). NMR studies showed the presence of a single bond C₁₆–C₁₇ (instead of the double bond in (1) and a tetrahydropyrane cycle due to the formation of C₁₃H–O–C₁₇H bond confirmed by a low field shift of C₁₃ (Δ∼10 p.p.m.) in comparison with ¹³C NMR spectrum of (1) (81.73 and 72.15 p.p.m. for C₁₃ in (1) and (2), respectively) (Table 1).² The tetrahydropyrane ring contains Br in position C₁₆. The spin coupling constants in the tetrahydropyrane ring correlate with ‘chair’ conformation and correspond to the following orientation of hydrogen atoms: C₁₃H_eq, C₁₄H_eq, C₁₆H_ax and C₁₇H_ax. On the basis of these coupling constant values and taking in mind that oligomycin A moiety has 13S and 14R configuration,³ we can ascribe 13S, 14R, 16R and 17S stereochemistry to bromooligomycin (2). This structure represents a new skeleton in this type of antibiotics.

Figure 1

Synthesis of the bromo derivative of 1 (compound 2).

Table 1 The ¹H and ¹³C NMR spectra of 1 and 2

The NMR spectra were elucidated using homo ¹H, ¹H (2D COSY) and hetero ¹H, ¹³C (2D HETCOR) correlation as well as ¹H and ¹³C 1D spectra of (1) and its derivatives and were registered using unity +400 (Varian, Palo Alto, CA, USA) spectrometer at 400.0 MHz for ¹H nuclei, and at 100.6 MHz for ¹³C nuclei.

The structure of (2) is membranous; a membranous structure has been demonstrated also for the product of the interaction of (1) with hydroxylamine.² It is likely that the formation of such structures is typical for this 26-membered macrolide as well as for 14-membered antibacterial macrolides (for example, erythromycin and its derivatives).⁴

Bromo-oligomycin A (2) was low cytotoxic against HCT116 human colon cancer cell line and K562 human leukemia cell line (IC₅₀ 16.0±2.2 and 9.0±1.4 μM, respectively), whereas the parental compound (1) was significantly more potent (IC₅₀ 3.3±1.0 and 3.0±1.0 μM, respectively) as determined by MTT-test after a 72 h incubation.⁵ Furthermore, (2) was weakly active against Streptomyces fradiae strain that is extremely sensitive to (1) (250 nM per disk and 0.001 nM per disk, respectively).

A lowered potency of (2) for actinobacterial cells correlated with a decreased activity against filamentous fungi (moulds) and yeast. Compound (1) was highly active against Aspergillus niger ATCC 16404 and Fusarium oxysporum VKM F-140 strains (MICs 0.125 and 1 μg ml⁻¹, respectively) and moderately active against Candida albicans ATCC 14053 and Cryptococcus humicolus ATCC 9949 (MICs 4 and 2 μg ml⁻¹, respectively, after a 24-h exposure). Compound (2) revealed the activity only against C. humicolus (MIC 2 μg ml⁻¹), whereas for other studied fungi and yeast strains (2) was practically inert (MIC >16 μg ml⁻¹). Determination of MICs was carried out according to NCCLS, Standards M27-A and M38-A.^{6, 7}

Low biological activities of compound (2) may be explained by distorted conformation of oligomycin A derivative in comparison with the starting antibiotic due to the formation of the webbed structure and also by the absence of free 13-OH group that participates at the interaction with oligomycin A target in cells. It was supposed that in oligomycins the hydroxyl groups could function as donors or acceptors of hydrogen bonds.⁸

Experimental procedure

Oligomycin A ((1); purity 95 %) was obtained in the Research Center for Biotechnology of Antibiotics BIOAN, Moscow using the Streptomyces avermitilis NIC B62 strain (production of (1) ∼1 g l⁻¹). Fermentation was performed for 8 days at 28 °C in liquid medium. Isolation and purification involved the extraction with acetone-hexane mixture followed by crystallization.

Synthesis of bromo-oligomycin A (2).

To a solution of (1) (1.0 g, 1.3 mmol) in dry DMF (10 ml) N-Br-succinimide (1-bromopyrrolidine-2,5-dione) (0.25 g, 1.41 mmol) was added, the reaction mixture was stirred at room temperature for 2 h and analyzed by TLC in CHCl₃-MeOH (10 : 0.5). The mixture was diluted with water; the reaction product was extracted with toluene, washed and dried over Na₂SO₄. Then the reaction product was purified by column chromatography on silica gel 60 (Merck) in CHCl₃-MeOH (50 : 0.5) to give 0.45 g (45%) of (2) as colorless amorphous powder. MW calcd. for C₄₅H₇₃BrO₁₁ 868.4336. Found in ESI-mass spectrum (m/z) 869.4445 (M + H)⁺, 871.4424 (M + H)⁺, 891.4274 (M + Na)⁺, 893.4265 (M + Na)⁺ (Bruker Daltonics GmbH, Bremen, Germany). UV-spectrum (λ_max nm, MeOH), (ɛ): 209 (14 000) (UV/VIS double beam spectrometer, UNICO, Dayton, NJ, USA); IR V_max, cm⁻¹ (film) 3417, 2967, 2934, 2877, 1698, 1644, 1456, 1365, 1277, 1224, 1172, 1092, 1046, 982, 881 and 763 (Nicolet_iS10 Fourier transform IR spectrometer, Nicolet, Madison, WI, USA); [α]_D²⁰ −83.3 (c 0.23, MeOH) (AA55 Polarimeter, Optical Activity Ltd (Cambridge, UK)); R_t 14.01 (Shimadzu LC10 vp instrument, Kyoto, Japan, MeCN-0.01 M H₃PO₄ mixture, pH 2.6. The percentage of MeCN increased from 20 to 22% within 15 min, then to 90% within 15 min at a flow rate of 1 ml min⁻¹).